Identification and prognostic biomarkers among ZDHHC4/12/18/24, and APT2 in lung adenocarcinoma

S-palmitoylases and S-depalmitoylases are differentially expressed in various cancers and several malignant tumors and show a strong prognostic ability. Notwithstanding, the potential clinical impact of S-palmitoylases and S-depalmitoylases, particularly in the prognosis and progression of lung adenocarcinoma (LUAD), has not been clarified. Expression levels of S-palmitoylases and S-depalmitoylases in LUAD were investigated using TCGA. GEPIA was used to evaluate the mRNA levels of S-palmitoylases and S-depalmitoylases at different pathological stages. Metascape was used to investigate the biological significance of S-palmitoylases and S-depalmitoylases. The Kaplan–Meier plotter was used to analyze the prognostic value of S-palmitoylases and S-depalmitoylases. CBioportal was used to analyze gene alterations in S-palmitoylases and S-depalmitoylases. UALCAN was used to examine DNA promoter methylation levels of S-palmitoylases and S-depalmitoylases. Finally, we investigated the relationship between S-palmitoylases, S-depalmitoylases, and tumor-infiltrating immune cells using TIMER. Correlations with immune checkpoint-related genes were determined using the R packages reshape2, ggpubr, ggplot2, and corrplot. PCR was also performed to assess the degree of ZDHHC4/12/18/24 and APT2 transcript expression in lung adenocarcinoma and adjacent normal lung tissues. HPA was utilized to investigate protein levels of S-palmitoylases and S-depalmitoylases in LUAD and normal lung tissue. Our study found that ZDHHC2/3/4/5/6/7/9/12/13/16/18/20/21/23/24, APT1/2, PPT1, LYPLAL1, ABHD4/10/11/12/13 and ABHD17C mRNA expression was significantly upregulated in LUAD, whereas ZDHHC1/8/11/11B/14/15/17/19/22, ABHD6/16A and ABHD17A mRNA expression was significantly downregulated. The functions of the differentially expressed S-palmitoylases and S-depalmitoylases were mainly associated with protein-cysteine S-palmitoyltransferase and protein-cysteine S-acyltransferase activities. Patients with high expression of ZDHHC4/12/18/24, APT2, ABHD4, ABHD11 and ABHD12 had a shorter overall survival. Infiltration of six immune cells (B cells, CD8+ T cells, CD4+ T cells, macrophages, neutrophils, and dendritic cells) was closely associated with the expression of ZDHHC4/12/18/24 and APT2. ZDHHC4/12/18/24 and APT2 positively correlated with the immune checkpoint-related gene CD276. We assessed the mRNA levels of ZDHHC4/12/18/24 and APT2 using qRT-PCR and found increased expression of ZDHHC4/12/18/24 in LUAD compared with healty control lung tissues. ZDHHC4/12/18/24, and APT2 are potential prognostic biomarkers of LUAD. Their expression levels could be related to the tumor microenvironment in LUAD.


Data collection and data processing
The Cancer Genome Atlas (TCGA) is a publicly financed effort that seeks to identify and classify significant genetic abnormalities associated with cancer to produce an extensive "atlas" of cancer genomic profiles 22 .The clinical characteristic data of patients with LUAD were downloaded from the TCGA database, which included 535 LUAD samples and 59 adjacent normal tissues.The specific characteristics included age, sex, smoking status, survival status, TNM stage, and pathological stage (Table 1).
Human specimens were obtained from the Department of Biobank, Division of Clinical Research, the First Hospital of Jilin University.Patients with LUAD who underwent surgery at the First Hospital of Jilin University participated in the study.Before surgery or cancer diagnosis, no patients had experience with chemotherapy, radiation, or antineoplastic therapy.Lung adenocarcinoma was histologically diagnosed in every patient.Lung cancer tissues were surgically removed, together with nearby normal tissues that were more than 5 cm distant from the tumor, and the tissues were stored at − 80 ℃.Age, gender, and TNM stage were among the clinical characteristics gathered.The AJCC Cancer Staging Manual, 8th Edition and the Veterans Administration Lung Cancer Study Group 3.30 criteria were used to establishing clinical stages.The Ethical Committee of the First Hospital of Jilin University approved our study.This study was conducted in accordance with the Declaration of Helsinki, and all patients provided written informed consent.

Correlations with transcriptional levels of target genes
S-palmitoylases and S-depalmitoylases RNA-sequencing expression (level 3) profiles and associated clinical data were retrieved from the TCGA dataset (https:// portal.gdc.com).The most recent version (V8) of the Genotype-Tissue Expression datas was downloaded from the Genotype-Tissue Expression project data portal website (https:// www.gtexp ortal.org/ home/ datas ets).We selected 513 lung adenocarcinoma samples from the TCGA database and 637 normal samples from the paracancerous and Genotype-Tissue Expression databases.Statistical

The correlation between targets of gene expression and pathological stages
The web-based application called Gene Expression Profiling Interactive Analysis(GEPIA) uses TCGA and Genotype-Tissue Expression data to provide rapid and adjustable functionality.Differential expression analysis, profiling visualization, correlation analysis, and patient survival analysis are just a few of the essential interactive and adaptable features offered by Gene Expression Profiling Interactive Analysis 23 .In this investigation, we carried out analyses of the association between pathology stages and expression of S-palmitoylases and S-depalmitoylases using the "LUAD" datasets in Gene Expression Profiling Interactive Analysis.A p value was calculated using Student's t-test, and the threshold p value was set at 0.05.

Creation of a protein-protein interaction network
STRING (http:// string-db.org; version 11.0) was used to evaluate the functional relationships between nodes and characterize the protein co-regulation of S-palmitoylases and S-depalmitoylases 24 .The default threshold of 0.4 was used to assess whether an interaction was statistically significant.An extensive gene list annotation and analysis resource for experimental biologists is offered by the web-based portal Metascape 25 .Gene ontology (GO) analysis, including biological process, cellular component, and molecular function enrichment analysis, was conducted for the selected S-palmitoylases and S-depalmitoylases using Metascape, then.Statistical significance was defined as a p value < 0.05.ClueGO (version 1.5.3) was used to assess and depict the functional enrichment of gene ontology, including biological processes, cellular components, molecular function, and Kyoto Encyclopedia of Genes and Genomes (KEGG): BP, CC, MF, and KEGG.

Survival analysis
The Kaplan-Meier plotter, an integrated database and online tool with multivariate and univariate analytic capabilities for in silico validation of novel biomarker candidates in NSCLC is available at www. kmplot.com/ lung 26 .In this study, to assess the overall survival(OS), first-progression survival(FP)-time to first progression, Table1.Characteristics of LUAD patients.

Associations between DNA promoter methylation and transcript expression level
UALCAN, an interactive, user-friendly online platform 28 was used to show the promoter methylation status of ZDHHC4/12/18/24, APT2,ABHD4,ABHD11,and ABHD12 in LUAD and healthy tissues.The p-value was calculated using Student's t-test, and the threshold was set at 0.05.

Immune infiltration analysis
The Tumor Immune Estimation Resource (cistrome.shinyapps.io/timer) is a platform for studying the connection between genes and tumor-infiltrating immune cells 29 .On the TIMER online website, we assessed the association between S-palmitoylases and S-depalmitoylases and B cells, CD4 + T cells, CD8 + T cells, dendritic cells, macrophages, and neutrophils.

Quantitative real-time polymerase chain reaction
TransZol (TransGen, CN) was used to collect the total RNA from the cells, and RNA concentration and purity were determined with BioTek Epoch 2 microplate Reader (Biotek, USA), after which cDNA was synthesized via reverse transcription using Hifair ® II 1st strand cDNA Synthesis SuperMix (Yeasen, CN).cDNA was used for subsequent quantitative real-time polymerase chain reaction (YEASEN, CN).A two-step process was performed using the 2XRealStar Green Fast Mixture with ROX II (GenStar, CN), with actin serving as the internal reference.The relative gene expression levels were calculated using the 2 -△△Ct method.The primer sequences are listed in the Supplementary Material (Supplementary 1. Table S1).

Protein-level analysis of target genes
The Human Protein Atlas is a library of protein expression patterns based on immunohistochemistry in various cancers, normal tissues, and cell lines 30 .This database was used to acquire S-palmitoylase and S-depalmitoylase expression using immunohistochemistry images from clinical specimens taken from patients with LUAD compared with healthy control lung tissues.

Predicted S-palmitoylation sites
GP S-Palm is a deep-learning-based graphic presentation system for the prediction of S-palmitoylation sites in proteins 31 .We speculated that CD276 may be palmitoylated by ZDHHC4/12/18/24 and affect tumor growth; therefore, we used this GP S-Palm software to predict CD276 palmitoylation sites.

Statistical analysis
GraphPad Prism (version 7.0) was used to evaluate and present the transcript expression patterns in LUAD patients obtained from clinical samples.In both LUAD and adjacent healthy lung tissues, we assessed the mRNA expression levels of ZDHHC4/12/18/24 and APT2.Differences were deemed significant at p < 0.05 following independent-sample Student's t-test analysis.

Aberrant expression of S-palmitoylases and S-depalmitoylases in patients with LUAD
The results showed that in LUAD, transcriptional levels of ZDHHC2/ was not statistically significant (Fig. 1).
To identify whether S-palmitoylases and S-depalmitoylases were associated with tumorigenesis, progression, and clinical outcome, we investigated the link between the expression of each S-palmitoylase and S-depalmitoylase and the pathological stage of LUAD (Supplementary 2 Figure S1).The mRNA expression of ZDHHC21 decreased as the tumor developed from stage I to stage II, suggesting a role for ZDHHC21 in the control of LUAD carcinogenesis and development.www.nature.com/scientificreports/

Enrichment analysis of S-palmitoylases and S-depalmitoylases
To investigate possible connections between differentially expressed S-palmitoylases and S-depalmitoylases, www.nature.com/scientificreports/we used STRING to conduct protein-protein interaction network analysis.In line with our expectations, the protein-protein interaction network produced multiple nodes of 38 and several edges of 232 (Fig. 2A).Gene ontology analysis in METASCAPE was used to anticipate the roles of S-palmitoylases and S-depalmitoylases (Supplementary 3 Figure S2).Regarding cellular components, the intrinsic component of the Golgi membrane was most highly enriched.In the molecular function category, protein-cysteine S-palmitoyltransferase, proteincysteine S-stearoyl transferase, and palmitoyl-(protein) hydrolase activities were enriched.In the biological processes category, lipoprotein metabolic processes, regulation of protein localization to the plasma membrane, and fatty acid derivative metabolic processes were significantly enriched.Functional annotations from ClueGO and CluePedia indicated a network of S-palmitoylases and S-depalmitoylases (Fig. 2B and 2C).The list of comprehensive functional notes and categorization pie charts were shown in Fig. 2D: 28.57% of terms belong to

Relationship between immune cell infiltration and S-palmitoylase and S-depalmitoylase expression in patients with LUAD
To investigate whether S-palmitoylases and S-depalmitoylases are effective biomarkers for immune therapy, we investigated the relationship between expression of S-palmitoylases and S-depalmitoylases and immune infiltration levels in LUAD.ZDHHC4 expression negatively correlated with B cells, CD8 + T cells, CD4 + T cells, macrophages, and neutrophils.ZDHHC12 expression was negatively associated with B cells, CD8 + T cells, macrophages, neutrophils, or dendritic cells.In addition, ZDHHC18 expression positively correlated with B cells, CD8 + T cells, CD4 + T cells, macrophages, neutrophils, and dendritic cells.ZDHHC24 expression was positively correlated with B cells and CD4 + T cells and negatively correlated with CD8 + T cells.APT2 expression negatively correlated with B cells, CD8 + T cells, CD4 + T cells, macrophages, neutrophils, and dendritic cells.And ABHD11 expression was negatively correlated with CD8 + T cells.However, ABHD4 and ABHD12 were not correlated with immune cell infiltration in LUAD.(Fig. 5).

ZDHHC4/12/18/24 expression is considerably higher in lung adenocarcinoma tissues
We found that the upregulation of ZDHHC4/12/18/24 and APT2 in LUAD and the high expression of ZDHHC4/12/18/24 and APT2 correlated with undesirable clinical consequences.Therefore, we intended to authenticate the mRNA expression levels of ZDHHC4/12/18/24 and APT2 in LUAD and compare them with normal lung tissues in vitro.We conducted a quantitative real-time polymerase chain reaction to compare the mRNA expression levels of ZDHHC4/12/18/24 and APT2 in LUAD and found that ZDHHC4/12/18/24 expression was significantly upregulated in LUAD.The APT2 expression level was not significantly different between LUAD and normal lung tissues (Fig. 7).

Discussion
The ZDHHC protein family contains a conserved protein domain consisting of 51 amino acids, and the domain contains a motif that is embedded in a cysteine-rich domain 32 .Recent genetic analysis in yeast showed that www.nature.com/scientificreports/ZDHHC proteins mediate palmitoylation 33 .Most members of the ZDHHC family are associated with many human diseases such as neuropsychiatric diseases and cancers 34 .The function of the ZDHHC family in cancer has been the subject of extensive research.For instance, several different types of cancers have been shown to have excessive ZDHHC3 expression, such as breast cancer 13 .Over the past decades, studies have demonstrated that ZDHHC8 siRNA and X-irradiation significantly slowed tumor development in 211H tumor-bearing mice 35 .Furthermore, ZDHHC9 has also been reported to be overexpressed in diverse types of cancers.Disruption of PD-L1 palmitoylation by preventing ZDHHC9 expression makes breast cancer cells more susceptible to T-cell-initiated death, which inhibits tumor development 36 .In previous reports, it was found that ZDHHC11 is overexpressed in NSCLC and high-grade bladder cancers 37,38 .In addition, we found that ZDHHC12 participated in apoptosis.Additionally, the BCL-2 associated X protein BAX, which is an essential component of the intrinsic apoptotic pathway of cell apoptosis 39 , maybe palmitoylated more frequently as a result of ZDHHC12 overexpression.Therefore, we hypothesize that ZDHHC12 might modify BAX palmitoylation to control tumor cell death, thereby affecting tumor cell proliferation.In addition, ZDHHC16 has good application prospects for improving the prognosis of hepatocellular carcinoma patients 40 .In gliomas, ZDHHC18 was upregulated and was associated with malignancy 41 .Previous studies have demonstrated that PPT1 is increased in cancer and is crucial for constraining tumor growth 18 .However, the expression of all S-palmitoylases and S-depalmitoylases in LUAD had not yet been comprehensively investigated.Therefore, a better understanding of the function of S-palmitoylases and S-depalmitoylases in LUAD was necessary for the future development of S-palmitoylases and S-depalmitoylases in LUAD therapy.
We also conducted quantitative real-time polymerase chain reaction assays to compare transcript expression levels of ZDHHC4/12/18/24 and APT2 in LUAD and found that ZDHHC4/12/18/24 expression was in accordance with TCGA analysis results.However, we may not have obtained exact findings because of the small sample size.Furthermore, the specimens collected from patients may exist in different pathological stages.We verified the expression of S-palmitoylases and S-depalmitoylases at the protein level, using the Human Protein Atlas database.We discovered that ZDHHC4/18/24 expression was noticeably higher in LUAD tissues than in normal tissues, however, ABHD4/11/12 protein level were decreased than in LUAD tissues (Supplementary 13 Figure S7).
In previous studies, differentially expressed S-palmitoylases and S-depalmitoylases have been found to affect patient survival.For example, we found that higher ZDHHC3 expression correlated with decreased survival in patients with breast cancer 13 .Based on the results of this study, we found a strong correlation between human ZDHHC8 expression and cancer survival 42 .Furthermore, genetic inhibition of S-depalmitoylase PPT1 suppresses tumor growth, and high PPT1 expression tends to aggravate the survival of patients with numerous distinct cancers 18 .In our study, according to the results from the Kaplan-Meier plotter, patients with LUAD with high transcript levels of ZDHHC4/12/18/24 and APT2 had a shorter overall survival time than those with low levels.www.nature.com/scientificreports/These data demonstrate that ZDHHC4/12/18/24 and APT2 in LUAD may predict survival outcomes.Moreover, numerous malignancies have been found to depend heavily on ZDHHC12 expression; for example, knockdown of ZDHHC12 inhibits the malignant CLDN3 accurate membrane localization and tumorigenesis of ovarian cancer 39 .
In glioblastoma, knockdown of ZDHHC12 restrains the growth, migration, and invasion capabilities 43 .In our study, poor overall survival in LUAD was linked to high expression of APT2.Therefore, APT2 expression may be an indicator of the prognosis of LUAD.As previously reported, APT1 and APT2 are significantly overexpressed in chronic lymphocytic leukemia cells.APTs directly interact with CD95 to accelerate depalmitoylation, which damages CD95-mediated apoptosis.CD95-mediated apoptosis in chronic lymphocytic leukemia cells can be reversed by treatment with miR S-138/-424 44 .Another study suggested that in Snail-transformed cells, blockade of APT2 recovers Scribble localization and S-palmitoylation 45 .Therefore, ZDHHC4/12/18/24 and APT2 may serve as biomarkers for predicting survival in patients with LUAD.Since S-palmitoylases and S-depalmitoylases varied markedly in LUAD, we examined the molecular features of LUAD.The main results of S-palmitoylases and S-depalmitoylases mutation were increased mRNA transcript expression.The mutation rates of all S-palmitoylases and S-depalmitoylases were low.On investigating the relationship between S-palmitoylase and S-depalmitoylase transcript expression levels and promoter methylation in LUAD, we found that the differentially expressed ZDHHC4/12/18/24 and APT2 were influenced by methylation levels, and expression levels of ZDHHC4/12 and APT2 were negatively correlated with the methylation level.These findings support earlier research that found a negative correlation between ZDHHC12 methylation and expression in glioblastomas 43 .
Immune cell infiltration plays a significant role in tumor progression and prognosis 46 .In this study, we found a significant correlation between the expression of ZDHHC4/12/18/24 and APT2 and the infiltration of the immune cells, suggesting that ZDHHC4/12/18/24 and APT2s are not only as prognostic indicators, but may also reflect immune status.In general, tumor-infiltrating cells, especially CD8 + T cells, have a good prognosis for NSCLC 47 .In LUAD, the expression of ZDHHC12/24 and APT2 was negatively correlated with CD8 + T cells.This suggests that ZDHHC12/24 and APT2 may inhibit tumor-associated CD8 + T cells, thereby aggravating the pathogenesis of LUAD and promoting tumor growth.We also investigated the correlation between immune checkpoint-related genes and the expression of ZDHHC4/12/18/24 and APT2.There was an evident positive association between the expression of the immune checkpoint-related gene CD276 and the expression of ZDHHC4/12/18/24 and APT2.The immune checkpoint-related gene, CD276, is commonly known as B7-H3.Previous research has shown that CD276 helps tumor cells evade the immune system in non-small cell lung cancer by collaborating with Tregs 48 .From several previous study, we had determined that CD276 might affect tumors via the JAK-STAT3 pathway [49][50][51] .In this study, GP S-Palm software identified three high-scoring palmitoylation sites in CD276 (Supplementary 14 Table S7).Therefore, we propose that CD276 might be palmitoylated, which would impact protein function.In this study, we demonstrated that ZDHHC4/12/18/24 and APT2 may affect CD276 palmitoylation and, therefore, tumor growth.
Our study has certain limitations.First, despite the high expression of ZDHHC4/12/18/24 and APT2 in LUAD as a strong prognostic factor for the survival of LUAD, almost all data used in our investigation were obtained from a web database.Therefore, further studies are required to confirm our findings and investigate the therapeutic use of S-palmitoylases and S-depalmitoylases in cancer treatment.Second, we did not explore the mechanisms of S-palmitoylases and S-depalmitoylases in LUAD.Future studies should explore these underlying mechanisms.Furthermore, ZDHHC4/12/18/24 and APT2 play crucial roles in the onset and progression of LUAD.These markers also have prognostic and diagnostic relevance.However, while this exhibits potential value, it is also uncertain, and further verification of the outcomes of los S-of-function and gain-of-function is required.

Figure 1 .
Figure 1.The transcriptional level of S-palmitoylases and S-depalmitoylases from the TCGA cohort.S-palmitoylases and S-depalmitoylases mRNA expression in LUAD tissues and normal tissues.The statistical differences between the two groups were determined by Student's t-test and the p value cutoff was 0.05.The left red band stands for LUAD group and the right blue band for the healthy control group.ns = not significant; * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 2 .
Figure 2. The enrichment analysis of S-palmitoylases and S-depalmitoylases in LUAD.(A) Protein-protein interaction network of S-palmitoylases and S-depalmitoylases.(B-D) The GO and KEGG functional annotation analysis of S-palmitoylases and S-depalmitoylases in LUAD was constructed using ClueGO, a plug-in of Cytoscape.The GO and KEGG enrichment analysis of the S-palmitoylases and S-depalmitoylases are shown in different colors.The results showed that significant differences in expression were related to protein-cysteine S-palmitoyltransferase activity and palmitoyl-(protein) hydrolase activity.

Figure 3 .
Figure 3. Overall survival analysis of ZDHHC4/12/18/24, APT2, ABHD4, ABHD11, and ABHD12 in LUAD patients.Patients were divided into two groups including the high (the red line) and low (the black line) groups expression based on ZDHHC4/12/18/24, APT2, ABHD4,ABHD11, and ABHD12 expression level.A log-rank test was used to estimate the difference in overall survival and a value of p < 0.05 was considered statistically significant.